MicroRNAs (miRNAs) are a group of non-coding RNA molecules with approximate 22 nucleotides in length and play an important role in the network of gene regulation. In body fluid, such as serum, plasma, saliva, urine and milk, etc., miRNAs exist highly stably outside of the cells. MiRNAs are associated to many diseases. The abnormal expressions of miRNAs, especially of circulating miRNAs have been detected under many pathological conditions. These pathological conditions include cancers, diabetes, heart failure, acute myocardial infarction and tissue damage, etc. Under certain pathological circumstances, in particular, with diseases such as cancers, the expression profile of miRNAs, especially the circulating miRNAs may significantly change according to the changes in physiological and pathological conditions. These researches have shown that miRNAs have great potential as non-invasive markers for molecular diagnosis and prognosis.
In recent years there have been several methods used for quantitative measurement of the miRNAs in clinical samples, such as Solexa sequencing, qRT-PCR and microarrays. The accuracy of the above-mentioned methods depends to a great extent on the reference gene(s) used. The reference gene(s) can be one gene or a combination of genes and shall be expressed stably under various experimental conditions and in different sample sets. In clinical application, detection of the miRNAs as biological markers must be normalized so that the detection process can be repeated in any lab. However, when quantitatively measuring the miRNAs, the changes of the amount of raw materials, the differences in sample collection and storage, and the efficiency differences in RNA extraction and enzymolysis may all lead to potential deviation and errors in quantification. In small size samples, the amount of total RNA is even smaller than the limits of the precise quantification by spectrophotometric method. All of the above-mentioned factors severely reduced the accuracy and reliability in quantitative analysis of miRNAs.
An ideal reference gene for miRNA normalization should meet the following requirements:
1) it is expressed stably in all samples and under all experimental conditions;
2) the amount of its expression can be compared to the study subjects;
3) it possesses the similar attributes of the study subjects, such as the stability and size of RNA, etc.
So far when the sample miRNAs are standardly measured, the selection of the reference gene relies still on experience, and the reference gene that meets the above-mentioned requirements has not been defined. In recent years artificially synthesized non-human (such as nematode) miRNAs have been used as exogenous control for normalized miRNAs detection. However, the exogenous controls are not the first choice because they can not correct the discrepancy in sample collection. Therefore, it is not an ideal reference gene.
Meanwhile, some endogenous genes have been frequently used as internal reference genes for the detection of tissue/cell miRNAs, such as 5SrRNA, 18SrRNA and U6 etc. Yet due to the fact that these genes are not miRNAs, they cannot represent the composition of miRNAs; and the efficiency of extraction, reverse transcription and PCR amplification of these genes might be different from that of the miRNAs. Therefore these genes are not the ideal choice, either.
Till present no research has been conducted on systematic characterization and evaluation on ideal reference gene(s) for miRNA normalization. Therefore, in this field it is necessary to develop the reference gene(s) for microRNA normalization and to establish an efficient normalization plan for the detection of miRNAs, especially circulating miRNAs.